In regard to biochemical and immunological investigations, there is a great need for detecting and measuring thin organic surface layers, in particular surface layers of that kind which have refractive indices of about 1.50 and which are of thicknesses in the range of from 1 to 10 nm. Such investigations are of particular interest in connection with toxin-receptor, enzyme-substrate or antigen-antibody reactions. It is known that an antigen-antibody reaction, that is to say, binding an antigen to its specific antibody, takes place even when one of the two reactants is bound to a surface of a substrate or a slide. It is therefore possible to bind a protein from a microorganism to a surface of a slide and immerse the slide in blood serum from a person. If that person is injected with a microorganism and the blood serum contains antibodies against the proteins of the microorganism, such antibodies will be bound to the protein which is present on the immersed surface of the slide if that protein is of the same type as the proteins of the microorganism. In that way, an organic layer is grown on the immersed surface. In the same way, it is obviously also possible for an antibody against a given protein to be bound to the immersed surface and used to detect the protein in the solution. Such investigation procedures have become increasingly important in recent years, particularly because monoclonal antibodies against a wide range of proteins can be produced by hybridoma techniques. An advantage of surface reactions, as compared to reactions in a liquid phase, is that there is only a low level of consumption of reagents.
However, the use of antibodies and antigens which are immobilised on surfaces or bound to surfaces in immunological investigations is hampered by the lack of simple and sensitive methods for measuring the thickness of the organic films, in the nm-range. An ellipsometer is used for that purpose in laboratory procedures. However, that is relatively expensive and can only be used by a skilled operator. Simplified investigation techniques which are based for example on the change in wettability, the scattering of light from metal particles and the sticking of colloidal particles have not been successful under practical conditions. Such simplified methods make use of the difference between a restricted area of the surface and the area around it. In such an operation, the restricted part of the surface is coated with an antigen or antibody, while the surrounding area is coated with another organic substance, in the same thickness. If proteins are bound in the restricted part of the surface, and not to the surface therearound, the thickness of the layer in the restricted part of the surface is greater than in the surrounding area. That gives different physical properties.
Antigen-antibody reactions and dielectric layers on reflecting carriers or substrates were the subject of the investigation a long time ago by Blodgett and Langmuir (Physical Review, volume 51, June 1937, pages 964 to 978).
Such investigations show that thin organic films on polished chromium surfaces can be observed if they are not applied directly to the naked surface but are applied to a barium stearate film which is formed on the chromium surface. Strong interference phenomena cannot be achieved, because the refractive indices are not of the optimum values, with a perpendicular angle of incidence. With angles of incidence, the interference phenomenon is greater, but in that case there are differences in the two polarisation states. It is therefore only possible to achieve substantial suppression of an individual wavelength, by simultaneously using a polarising means. When the sample is viewed through a polarising means, and by measuring the angle of incidence at minimum intensity, it is possible to determine the thickness of monomolecular films, in accordance with Blodgett and Langmuir, with a relatively high degree of accuracy, and thus detect a binding reaction between antigens and antibodies on surfaces. Immunological reactions have been investigated in this case, using a rudimentary ellipsometry process.
The use of substantial interference phenomena of tantalum oxide on tantalum has also been the subject of publication by Vroman (Thromb. Diath, Haemorrhag, volume 10, pages 455 to 493 (1964), in particular page 463).
Anodized tantalum plates with a dark brown colouring change towards violet if they are coated with a monomolecular protein layer. The oxide forms an anti-reflection coating on the metal and the plate has a brown appearance if the reflection of blue and green light is suppressed to a greater extent than the reflection of longer wavelengths. When a thin layer of protein is formed on the surface, the thickness of the transparent layer increases and the reflection minimum moves towards longer wavelengths. The reflected light therefore contains more blue and violet components, if a biological layer, for example a layer of protein, is present.
The same properties as were found by Vroman were also attained by Giever and Laffin, when a gold-indium alloy was applied to a glass slide and gradually oxidized so as to produce a brownish colour (The Journal of Immunology, volume 110, No 5, May 1973, pages 1424 to 1426; Biomedical Applications of Immobilized Enzymes and Proteins, volume 2, pages 147 to 162).
The last three known processes mentioned made use of the effect that thin biological layers, for example protein layers, which are applied to dielectric layers of a given thickness, alter the interference phenomena of the original dielectric layer. The known processes involve using metals which, when they are used as substrates, require thereon films which have a high refractive index if good interference properties are to be achieved, although a low level of optical detection sensitivity is tolerated. There is also the danger that the reactants might be affected by contact with the metal or metal oxides. That is an undesirable situation, particularly when the substance to be detected is present in the solutions in only low levels of concentration, and long incubation periods are required. Metal substrates, or metal oxide layers disposed thereon, also have indefinite surface properties as the surface energy and the surface density of the binding locations change and therefore the binding effect or the adsorption of organic molecules cannot be controlled with sufficient accuracy in that respect.
U.S. Pat. Nos. 3,926,564 and 3,979,184 also disclose metal systems for rendering thin surface layers visible. In U.S. Pat. No. 3,979,184, a dielectric layer is disposed between a metal substrate and a semi-transparent metal layer, while U.S. Pat. No. 3,926,564 provides that an oxide is formed on a noble metal alloy having an oxidizable component. Those processes also involve metal systems in which, as already mentioned above, a low level of optical detection sensitivity is tolerated.
It is also known (see Laurell, C.B. `Quantitative estimation of protein by electrophoresis in agarose gel containing antibodies`, Anal. Biochem. 15: 45, 1966), for the substance to be detected, or the amount thereof, to be allowed to spread by electrophoresis in a gel which contains a homogeneously distributed concentration of a reactant which reacts with the substance to be detected. The two reactants react by precipitation in the gel, and the reaction region is rendered visible, the reaction region being limited to the area in which the reactant for the substance to be detected is contained from the outset. That system provides quick and accurate detection. However, that detection method is subject to the restriction that the reactant which is used for the detection operation should not migrate in the electrical field, thereby giving rise to limitations in regard to the electrophoresis conditions such as for example the pH-value, the gel quality and the polarity involved. In addition, that method is restricted to those reactions which result in a precipitation effect.
In another known method (see Elwing, H. and Nygren, H. `Diffusion in gel-enzyme linked immunosorbent assay (DIG-ELILSA): A simple method for quantification of class-specific antibodies`, J. Immun. Methods, 31:101, 1979), the substances to be detected spread out by diffusion in a gel which is disposed above a surface provided with a thin layer of a reactant for detecting the substance in question. After a certain period of time, the gel is removed and the reaction region on the surface is rendered visible. The size of the reaction region is measured and constitutes a measurement in respect of the amount of the substance to be detected, which is diffused over the surface. The step of rendering the reaction product at the surface visible can be performed by secondary reactions such as by incubation with an isotope or enzyme-labelled antibodies directed against the reaction product bound after the diffusion process.
It is also possible to perform the detection operation by a water condensation process (Adams, A.L., Klings, M., Fischer, G.C. and Vroman, L., `Three simple ways to detect antibody antigen complex on flat surfaces`, J. Immun. Methods, 3:227, 1973). In addition, the reaction products may be rendered visible by direct optical analysis of the primary reaction product, by ellipsometry (Elwing, H. and Stenberg, M. `Biospecific bimolecular binding reactions--a new ellipsometric method for their detection, quantification and characterization`, J. Immun. Methods, 44:343, 1981) or by light interference on thin layers (Adams, A.L., Klings, M., Fischer, G.C. and Vroman, L., `Three simple ways to detect antibody antigen complex on flat surfaces`, J. Immun. Methods, 3:227, 1973). In rendering the reaction product visible, the advantage of a high level of sensitivity is attained by means of intensification reactions such as for example by using enzyme-labelled antibodies, while the optical detection methods provide direct detection of the primary reaction product. In addition, the optical detection methods do not use any reactant other than the reactant required for detection purposes.
In the diffusion method in which a surface is coated with a reactant for detecting a substance, there is the advantage, in comparison with the passive or active transport reaction of a reactant which is distributed in a gel, that it is also possible to investigate or detect reactions which do not result in a precipitation effect. In order to immobilise the reactant on the surface, there are a number of possible ways of binding the molecules of the reactant which is to be used for detecting the other substance. However, the diffusion methods involve a relatively long diffusion time. However, with the method disclosed in FEBS Letters, volume 135, No 1, November 1961, pages 73 to 76, it is possible to cause the substance to be detected to react with the reactant present on the surface of the substrate, by a binding action, in a relatively short period of time and at low cost. In addition, there are no limitations in regard to the electrophoresis conditions. However, difficulties do arise in regard to simplifying the step of rendering visible the substance to be detected, which is bound on the binding agent present on the surface of the substrate. There is also the danger, when using metal substrates, of undesirable electrochemical reactions, for example the formation of oxides on the substrate surface.
Biomedical methods of detecting organic substances in solutions are also known from German laid-open application (DE-OS) No. 25 12 730 and U.S. Pat. No. 4,181,501. The former specification discloses radial diffusion of the substance to be detected, in a gel, and transportation of that substance by means of electrophoresis in a gel, but the step of detecting such substances is performed by means of metal plates in regard to which, as already referred to above, a relatively low level of sensitivity has to be tolerated.